US10527549B2ActiveUtilityA1
Cross-talk correction in multiplexing analysis of biological sample
Est. expirySep 22, 2035(~9.2 yrs left)· nominal 20-yr term from priority
B01L 2300/0627G01N 2021/6441G01N 21/6452G01N 21/6428G01N 2021/058G01J 3/4406G01N 21/6456G01N 21/6408B01L 3/502715
85
PatentIndex Score
23
Cited by
15
References
13
Claims
Abstract
A method for determining fluorescence values {φispe}i∈{1, 2, . . . , I} of a set of I fluorescent microparticles {μPi}i∈{1, 2, . . . , I} of a multiplexed analysis, the microparticles being in a monolayer arrangement, includes acquiring a digital fluorescence image of the set of fluorescent microparticles {μPi}i∈{1, 2, . . . , I}; and computing, for each fluorescent microparticle μPi in the set of fluorescent microparticles {μPi}i∈{1, 2, . . . , I}, a fluorescence value φimeas based only on pixels of the acquired image corresponding to said fluorescent microparticle μPi. The method includes computing the fluorescence value φispe of the fluorescent microparticle μPi by correcting its first fluorescence φimeas by a cross-talk fluorescence contribution φicross in the first fluorescence φimeas from other fluorescent microparticles {μPj}j≠i in the set of fluorescent microparticles {μPi}i∈{1, 2, . . . , I}.
Claims
exact text as granted — not AI-modifiedThe invention claimed is:
1. A method for determining fluorescence values {φ i spe } i∈{1, 2, . . . I} of a set of I fluorescent microparticles {μP i } i∈{1, 2, . . . I} of a multiplexed analysis configured to detect multiple fluorescently labeled biomarkers, said microparticles being in a monolayer arrangement and encoded in silicon micro-disks carrying a binary code, the method comprising:
acquiring a digital fluorescence image of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . I} ;
computing, for each fluorescent encoded microparticle μP i in the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . I} , a fluorescence value φ i meas based only on pixels of the acquired image corresponding to said fluorescent encoded microparticle μP i , and
obtaining the fluorescence value φ i spe of said fluorescent encoded microparticle μP i using each fluorescence values φ i meas ;
wherein the method comprises computing the fluorescence value φ i spe of said fluorescent encoded microparticle μP i by correcting its first fluorescence φ i meas by a cross-talk fluorescence contribution φ i cross , wherein the cross-talk fluorescence contribution φ i cross is modeled as a sum of individual contributions, each having an isotropic decay profile:
φ
i
cross
=
∑
j
≠
i
(
α
i
j
·
φ
j
spe
)
wherein:
α ij is a unitary cross-talk fluorescence contribution in the first fluorescence φ i meas of the j th fluorescent microparticle μP j depending only on the distance d i,j between microparticles μP i and μP i and predetermined parameters that are identical whatever the microparticles,
the j th set of encoded microparticles is a subset created from the set of encoded microparticles {μP i }iε{1, 2, . . . , I} other than μP i , and
φ i spe is the fluorescence of the j th fluorescent encoded microparticle μP j .
2. A method as claimed in claim 1 , wherein the computation of the fluorescence value φ i spe comprises:
computing a position X i in the digital fluorescence image for each fluorescent encoded microparticle μP i in the set of fluorescent encoded microparticles {μP i } i∈{1, 2 . . . , I} ;
modelling the first fluorescence value φ i meas as a function of the positions {X i } i∈{1, . . . , I} and fluorescence values {φ i spe } i∈{1, . . . I} of all fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I}; and
computing the inverse of said function to obtain the fluorescence value φ i spe .
3. A method as claimed in claim 1 , wherein the method comprises:
computing the distance d i,j between the i th and the j th fluorescent encoded microparticles in the digital fluorescence image;
for each couple of encoded microparticles (μP i , μP j ) in the set of I fluorescent encoded microparticles, computing the unitary cross-talk fluorescence contribution α ij of said couple (μP i , μP j ) based on the distance d i,j ;
computing the fluorescences {φ i spe } i∈{1, 2, . . . , I} of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} based on the following relationship:
(
φ
1
spe
φ
2
spe
⋮
φ
I
-
1
spe
φ
I
spe
)
=
(
1
α
12
⋯
α
1
(
I
-
1
)
α
1
I
α
21
1
⋯
α
2
(
I
-
1
)
α
2
I
⋮
⋯
⋱
⋯
⋮
α
(
I
-
1
)
1
α
(
I
-
1
)
2
⋯
1
α
(
I
-
1
)
I
α
I
1
α
I
2
⋯
α
I
(
I
-
1
)
1
)
-
1
(
φ
1
meas
φ
2
meas
⋮
φ
I
-
1
meas
φ
I
meas
)
.
4. A method as claimed in claim 1 , wherein each fluorescent encoded microparticle μP i of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} comprises an identifier Id m (i) of a set of M different unique identifiers {id m } m∈{1, 2, . . . , M} , said identifiant Id M (i) being readable through processing of a digital image of said fluorescent encoded microparticle μP i , and in that the method further comprises:
acquiring a digital image of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} ;
reading the identifier Id m (i) of each encoded microparticle μP i in the digital image; and
for each identifier Id m of the set of M different unique identifiers {id m } m∈{1, 2, . . . , M} , computing an aggregate fluorescence φ m ag based on the fluorescences φ i spe of the fluorescent encoded microparticles comprising said identifiant.
5. A method as claimed in claim 4 , wherein each fluorescent encoded microparticle μP i of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} comprises a surface coated with fluorescent complexes uniquely associated to the identifier Id m (i) of said fluorescent encoded microparticle μP i , said complexes comprising first non-fluorescent molecules fixed to the encoded microparticles and second fluorescent molecules bound to the first non-fluorescent molecules.
6. A method as claimed in claim 5 , wherein the encoded microparticles have equal dimension.
7. A method as claimed in claim 5 , wherein the method comprises:
prior to acquiring the digital fluorescence image of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} ;
disposing in a channel the microparticles without any second fluorescent molecules bound to the first non-fluorescent molecules, so as to arrange the microparticles in a monolayer; and
filling the channel with a liquid sample,
computing concentration of second fluorescent molecules in the sample based on the aggregate fluorescences φ m ag .
8. A system for determining fluorescences {φ i spe } i∈{1, 2, . . . , I} of a set of I fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} being encoded in silicon micro-disks carrying a binary code comprising:
at least one channel for receiving the set of I fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} in a monolayer arrangement;
an acquisition unit for acquiring a digital fluorescent image of the monolayer arrangement of the set fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} in the channel;
a computing unit for computing the fluorescences {φ i spe } i∈{1, 2, . . . , I} based on the acquired digital fluorescent image, the computing unit computing a first fluorescence φ i meas based only on pixels of the acquired digital fluorescent image corresponding to said fluorescent encoded microparticle μP i , said computing unit being configured to obtain the fluorescence value φ i spe of a fluorescent encoded microparticle μP i using each fluorescence values φ i meas ;
wherein the computing unit computes, for each fluorescent encoded microparticle μP i in the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} :
a cross-talk fluorescence contribution φ i cross , wherein the cross-talk fluorescence contribution φ i cross is modeled as a sum of individual contributions, each having an isotrpic decay profile, based on the following relationship:
φ
i
cross
=
∑
j
≠
i
(
α
i
j
·
φ
j
spe
)
wherein:
α ij is a unitary cross-talk fluorescence contribution in the first fluorescence φ i meas of the j th fluorescent microparticle μP j depending only on the distance d i,j between microparticles μP i and μP j and predetermined parameters that are identical whatever the microparticles,
the j th set of encoded microparticles is a subset created from the set of encoded microparticles {μP i }iε{1, 2, . . . , I} other than μP i , and φ j spe is the fluorescence of the j th fluorescent encoded microparticle μP i ; and
the fluorescence φ i spe of said fluorescent encoded microparticle μP i by correcting its first fluorescence φ i meas by the cross-talk fluorescence contribution φ i cross .
9. A system as claimed in claim 8 , wherein the computing unit computes the fluorescence value φ i spe by:
computing a position X i in the digital fluorescence image for each fluorescent encoded microparticle μP i in the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} ;
modelling the first fluorescence value φ i meas as a function of the positions {X i } i∈{1, . . . , I} and fluorescence values {φ i spe } i∈{1, . . . , I} of all fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} ; and
computing the inverse of said function to obtain the fluorescence value φ i spe .
10. A system as claimed in claim 8 , wherein the computing unit:
computes the distance d i,j between the i th and the j th fluorescent encoded microparticles in the digital fluorescence image;
for each couple of encoded microparticles (μP i , μP j ) in the set of I fluorescent encoded microparticles, computes the unitary cross-talk fluorescence contribution α ij of said couple (μP i , μP j ) based on the distance d i,j ;
computes the fluorescences {φ i spe } i∈{1, 2, . . . , I} of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} based on the following relationship:
(
φ
1
spe
φ
2
spe
⋮
φ
I
-
1
spe
φ
I
spe
)
=
(
1
α
12
⋯
α
1
(
I
-
1
)
α
1
I
α
21
1
⋯
α
2
(
I
-
1
)
α
2
I
⋮
⋯
⋱
⋯
⋮
α
(
I
-
1
)
1
α
(
I
-
1
)
2
⋯
1
α
(
I
-
1
)
I
α
I
1
α
I
2
⋯
α
I
(
I
-
1
)
1
)
-
1
(
φ
1
meas
φ
2
meas
⋮
φ
I
-
1
meas
φ
I
meas
)
.
11. A system as claimed in claim 8 , wherein each fluorescent encoded microparticle μP i of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} comprises an identifier Id m (i) of a set of M different unique identifiers {id m } m∈{1, 2, . . . , M} said identifiant Id M (i) being readable through processing of a digital image of said fluorescent encoded microparticle μP i , and in that the computing unit:
acquires a digital image of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} ;
reads the identifier Id m (i) of each encoded microparticle μP i in the digital image; and
for each identifier Id m of the set of M different unique identifiers {id m } m∈{1, 2, . . . , M} computes an aggregate fluorescence φ m ag based on the fluorescences φ i spe of the fluorescent encoded microparticles comprising said identifiant.
12. A system as claimed in claim 11 , wherein each fluorescent encoded microparticle of the set of fluorescent encoded microparticles {μP i } i∈{1, 2, . . . , I} comprises a surface coated with fluorescent complexes uniquely associated to the identifier Id m (i) of said fluorescent encoded microparticle said complexes comprising first non-fluorescent molecules fixed to the encoded microparticles and second fluorescent molecules bound to the first non-fluorescent molecules.
13. A system as claimed in claim 12 , wherein the encoded microparticles have equal dimension.Cited by (0)
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